Solution of carbon monoxide for the treatment of disease, including sickle cell disease

ABSTRACT

The invention provides compositions and methods for delivering carbon monoxide (CO) to subjects suffering from inflammatory, cardiovascular, Sickle Cell, and other disease. The compositions are liquids, including Newtonian and non-Newtonian liquids, such as pastes, gels, foams, emulsions, and other non-gaseous compositions, in which CO is dissolved at an amount that, when administered to a patient, provides a therapeutically or prophylactically effective amount of CO to the patient. The compositions can be provided in many forms, including in bottles or cans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/979,510, filed Jul. 12, 2013, which was filed pursuant to 35 U.SC. §371 as a United States National Phase Application of InternationalApplication No. PCT/US2012/020710, filed Jan. 10, 2012, which claims thebenefit of the filing date of U.S. Provisional Application No.61/434,639, filed Jan. 20, 2011, and U.S. Provisional Application No.61/432,843, filed Jan. 14, 2011, all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the fields of therapeutics and medicalcompositions. More specifically, the invention relates to methods oftreatment for genetic, inflammatory, and other diseases that can betreated with therapeutic levels of carbon monoxide, including sicklecell disease, and to formulations and delivery vehicles that are usefulin performing the methods.

Description of Related Art

At the present time, methods and devices for treatment of Sickle CellCrises (referred to herein as “SCC”) and its prevention are not adequateto manage the disease. The U.S. Centers for Disease Control andPrevention has estimated that 70,000 to 100,000 patients suffer fromSickle Cell Disease (referred to herein as “SCD”) in the U.S., and thelife expectancy of these afflicted individuals is only about 40 years.When a patient is admitted to hospital, treatment is symptom-based,mainly by way of morphine-analogue analgesics, fluid replacement,transfusions, and other supportive measures. Currently, the onlyconsistent and reliable method of treatment of SCC is through bloodtransfusion. However, this treatment method is expensive andinconvenient, and can be dangerous. The use of hydroxyurea to preventcrises has been found to have marginal efficacy and to have sideeffects, and the response per patient is highly variable. It is alsopossible to cure SCD through bone marrow transplant, but this procedureis rarely used due to its inherent danger, its high cost, and theco-morbidities of the necessary treatment.

It is known that carbon monoxide (CO) is a poison at highconcentrations, interfering with the ability of red blood cells to carryoxygen. It has been reported that hemoglobin (referred herein as “Hb”)saturations of over 67% likely result in CO-induced death unlesstreatment is provided. Furthermore, Hb saturations of over 30% arereported to result in loss of consciousness, among other seriousmorbidities, and can result in death if maintained long-term. Inaddition, Hb saturations between 16% and 20% are reported to result inheadache and visual evoked response abnormalities (Stewart RD. Theeffect of carbon monoxide on humans. Annu Rev Pharmacol 15: 409-423,1975). As such, extreme caution must be used in situations where CO ispresent.

However, it is possible that CO can have positive effects in SCD and inother diseases and disorders. For example, it has been reported that COextends the red cell life span in SCD patients. (Beutler, E., 1975, “Theeffect of carbon monoxide on red cell life span in sickle cell disease.”Blood 46(2): 253-9.) It has also long been hypothesized that CO mightplay a role in preventing sickle cell formation by preventing thepolymerization of hemoglobin (Sirs, J. A., 1963, “The use of carbonmonoxide to prevent sickle cell formation”, Lancet 1, 7288: 971-2).Further, it has been reported that CO might have a preventative effecton the occurrence of clinical symptoms of SCD. (Yallop, D., E. R.Duncan, et al., 2007, “The associations between air quality and thenumber of hospital admissions for acute pain and sickle-cell disease inan urban environment.” Br J Haematol 136(6): 844-8.) The Yallop studydocuments that there is a decrease in hospital admissions of patientswith SCC on days with higher CO content in the breathed air.

Recent research has also found that CO can have more widespread healthbenefits in multiple diseases and organ systems, including incardiovascular, kidney, liver, lung, and intestine (Inge Bauer andBenedikt H J Pannen, “Bench-to-bedside review: Carbon monoxide—frommitochondrial poisoning to therapeutic use”, Critical Care 2009,13:220). Other research points to positive effects in inflammatory andcardiovascular disease (Foresti R, Bani-Hani M G, Motterlini R., “Use ofcarbon monoxide as a therapeutic agent: promises and challenges”,Intensive Care Med. 2008 April; 34(4):649-58. Epub 2008 Feb. 20).

In view of the proposed beneficial effects of CO on certain diseases anddisorders, a number of efforts using different delivery mechanisms havebeen made to employ CO as a treatment for disease. These include:delivery of CO gas via pulmonary delivery (Motterlini, R., Otterbein L.,“The therapeutic potential of carbon monoxide”, Nat Rev Drug Discov.2010 September; 9(9):728-43); the delivery of CO bound to a non-ferrousmetal in a small molecule via intravenous infusion, intra-peritonealinjection, or oral ingestion (Motterlini, R., Otterbein L., “Thetherapeutic potential of carbon monoxide”, Nat Rev Drug Discov. 2010September; 9(9):728-43); and the delivery of CO bound to a chemicallymodified human or bovine hemoglobin tetramer via intravenous infusion(Vandegriff, K. D., M. A. Young, et al. (2008). “CO-MP4, a polyethyleneglycol-conjugated haemoglobin derivative and carbon monoxide carrierthat reduces myocardial infarct size in rats.” Br J Pharmacol 154(8):1649-61; United States patent application publication number 20100311657Abuchowski, Abraham et al. “HEMOGLOBIN COMPOSITIONS” Dec. 9, 2010; andUnited States patent application publication number 20090082257 Winslow,Robert M. “MalPEG-Hb conjugate-containing compositions for deliveringcarbon monoxide (CO) to cells” Mar. 26, 2009). However, these effortsface a number of significant problems and shortcomings.

With regard to delivery of CO gas via inhalation, a number of problemsexist that have precluded its clinical use. One of the primary reasonsfor the lack of clinical use relates to the importance of dosage in COadministration. The efficacious dose of CO is relatively close to itstoxic dose. This makes pulmonary delivery difficult given differences inlung function in various diseases, including in SCD. A secondcomplication is that CO is excreted through the lungs. As such,pulmonary delivery of CO requires uptake and excretion through the sameorgan, significantly complicating pharmacokinetics and determinations ofsafety. Another challenge with pulmonary delivery is that pulmonarydelivery is inconvenient for patients given the discomfort of utilizinga breathing apparatus and the restriction on patient mobility given theneed to be close to the breathing apparatus during dosage periods. Thisis a potentially significant matter, as inconvenience for patients ishighly correlated to a lack of patient compliance. Moreover, theinherent toxicity of CO and its odorless, colorless properties makepulmonary delivery use challenging. Storing the amount of CO that wouldbe needed to treat a patient long-term could, in the case of the home,put the patient and other family members in danger, and, in the case ofthe hospital, would require novel and costly safety precautions such asmonitoring and venting before use, and even with such safeguards couldput hospital staff in danger.

The utilization of small molecule transition metal-based carriers of CO(referred to herein as Carbon Monoxide Releasing Molecules or “CORMs”)also presents significant challenges for clinical deliver of CO. Inlinking carbon monoxide to a transition metal, the toxicity of thetransition metal is added to the inherent toxicity CO. This transitionmetal toxicity can limit the acceptable dose and, for certain metals,prevents use in humans completely. Ruthenium and Molybdenum are two ofthe more widely used transition metals in forming CORMs, and thesemetals have been categorized as metals of significant safety concern bythe European Medicines Agency (EMEA COMMITTEE FOR MEDICINAL PRODUCTS FORHUMAN USE (CHMP), “GUIDELINE ON THE SPECIFICATION LIMITS FOR RESIDUES OFMETAL CATALYSTS OR METAL REAGENTS”, London, 21 Feb. 2008, Doc. Ref.EMEA/CHMP/SWP/4446/2000). This high potential toxicity of CORMs due tothe transition metal carriers prevents the use of CORMs in certainindications due to potentially toxicity-limited dosage and also througha more difficult risk:benefit ratio due to the added risk of thetransition metal. Particularly unstable patients, including SCDpatients, can be particularly at risk. In addition, the toxicity oftransition metal carriers presents a significant barrier to recurrentuse of CORMs in chronic indications. As SCD is an inherited lifelongcondition, long term use of CORMs as a therapy for prevention of SCC isunlikely to be safe as the transition metal carriers will accumulateover time, aggravating the potential toxicity. In summary, the use oftransition metal compounds as CO carriers has serious drawbacks ascompared to less toxic approaches.

The use of chemically modified hemoglobin tetramers as carriers of CO(cell free CO-Hb) also presents toxicity-related issues. It has beendemonstrated that certain significant safety events are associated withthe clinical use of hemoglobin tetramer-based oxygen carriers, includingmyocardial infarction and death, among others (Natanson C, et. al.“Cell-free hemoglobin-based blood substitutes and risk of myocardialinfarction and death: a meta-analysis”, JAMA. 2008 May 21;299(19):2304-12). The potential toxicity of cell free CO-Hb due to theuse of cell free hemoglobin as a CO carrier prevents the use of CO-Hb incertain indications due to potentially toxicity-limited dosage and alsothrough a more difficult risk:benefit ratio due to the added risk of thehemoglobin tetramer. Particularly unstable patients, including SCDpatients, can be particularly at risk. In addition, the toxicity of cellfree Hb in addition to the potential problematic iron load presents asignificant barrier to recurrent use of CO-Hb in chronic indications,including for use in prevention of SCC.

In addition, it has long been known that CO, as most gases, is solubleat low levels at ambient pressure in aqueous solutions. Solutions havepreviously been prepared in academic laboratories to demonstrate thisfact. In addition, aqueous solutions have previously been prepared atambient pressure and between 4° C. and 21° C., and used ex vivo innon-human studies in order to determine whether delivery of CO by suchsolutions could improve outcomes in the transplantation of gut, liver,and lung tissues (Nakao A et. al. “Ex vivo application of carbonmonoxide in University of Wisconsin solution to prevent intestinal coldischemia/reperfusion injury”, Am J Transplant. 2006; 6(10):2243-2255;Ikeda, A et. al. “Liver graft exposure to carbon monoxide during coldstorage protects sinusoidal endothelial cells and emelioratesreperfusion injury in rats”, Liver Transpl. 2009 November; 15(11):1458-1468; Nakao A et. al. “Ex vivo carbon monoxide prevents cytochromeP450 degradation and ischemia/reperfusion injury of kidney grafts”,Kidney International. 2008; 74:989-991). One study also looked at usingsuch a solution prepared at room temperature and pressure and injectedintraperitonealy (referred to herein as “IP”) to investigate whethersuch a solution could ameliorate postoperative ileus in mice (AtsunoriN, et. al., “A Single Intraperitoneal Dose of Carbon Monoxide-SaturatedRinger's Lactate Solution Ameliorates Postoperative Ileus in Mice”, JPET319:1265-1275, 2006). However, the use of this solution was severelylimited. First, in preparing the solution at room temperature andpressure, the amount of dissolved CO was very low. This preparationmethodology was necessary in this case because injecting a cold solutioncould be harmful if directly injected into the peritoneum and, moreover,as the liquid warmed, the CO would bubble out of the solution into theperitoneum which likely would cause potentially severe complications. Inaddition, while IP delivery is used in non-human research, it is rarelyused in treating human disease for both safety and convenience reasons.First, the potential for infection in IP injection is significant, whichcreates an additional risk to this delivery route. In addition, theinconvenience for patients due to IP delivery can correspond to a lackof patient or physician compliance. Also, in order to allow home IPinfusion, a permanent access into the peritoneal space would have to beplaced in the patient, similar to that used in IP dialysis. This wouldadd significant inconvenience and also potential morbidities, such asrisk of infection, as compared to a non-IP delivery route. Moreover, theIP delivery route relies upon provision of a small amount of CO locally,using direct delivery to the gastrointestinal tract, which is inherentlylimiting with regard to the treatment of disease.

In summary, to date, there has been no widely suitable, convenient, andsafe method for delivery of CO in amounts that would be therapeutic totreat diseases and disorders while avoiding toxicity and providing thenecessary level of convenience to those in need.

SUMMARY OF THE INVENTION

The present invention provides a new way to treat disease, includingcardiovascular, inflammatory, Sickle Cell, and other diseases for whichCO can be used therapeutically. The present invention provides a methodwhereby CO is dissolved or entrapped in a liquid and the liquid isadministered to a patient through the gastrointestinal (GI) tract orintravenously (IV) in an amount that delivers a treatment-effectiveamount of CO to the patient. The method of the invention safelyadministers CO to the afflicted patient, and provides a solution to along-felt need in the art for a safe treatment method and deliveryvehicle for CO. As used herein, the term “liquid” is given its broadestreasonable meaning, and thus includes both Newtonian liquids andnon-Newtonian liquids. It thus includes compositions in which the maincomponent, by weight, is a liquid. The term thus includes pastes, gels,and emulsions. It likewise includes foams in which CO bubbles areentrapped. For ease of reference, within the present disclosure,reference is made to “liquids” in which CO is “dissolved”. However, itis to be understood that such references are not limited to Newtonianliquids in which CO is in solution, but rather to all liquids, pastes,gels, emulsions, foams, etc. in which gaseous CO is dissolved,entrapped, etc. It is believed that this is the first report of in vivodelivery of a CO-containing liquid through the GI tract or IV fortherapeutic or prophylactic purposes. The invention allows for deliveryof set (i.e., predetermined) amounts of CO to a patient systemically totreat disease. The CO-containing liquid is, in exemplary embodiments,delivered orally or by injection or infusion.

In a first aspect, the invention provides a liquid compositioncontaining CO dissolved in an amount sufficient for delivery of the COto a patient (used herein interchangeably with “subject” and “person inneed”), wherein delivery to the patient results in an effectivetreatment on a disease or disorder that is treatable with CO. UnlikeCO-containing liquid compositions known in the art, the liquidcomposition according to the present invention has a CO concentrationthat is sufficiently high that an effective amount of CO can bedelivered to the patient in a convenient volume of liquid. That is,CO-containing liquid compositions known in the art, such as those knownfor investigating the effect of CO on surgically altered tissues, haverelatively low concentrations of CO, and are not suitable for in vivodelivery of CO to treat diseases and disorders. In contrast, the liquidcomposition of the present invention comprises a relatively highconcentration of CO, and can be used for in vivo delivery oftreatment-effective amounts of CO to patients. While not so limited, ingeneral, the composition of the present invention comprises awater-based composition in which CO is dissolved. Preferably, the CO isin gaseous form in solution, and is not complexed with a metal or one ormore Hb molecules or complexes. In exemplary embodiments, the liquidcomposition is a beverage, such as one provided in a sealable container.

In another aspect, the present invention provides a method for treatinga patient suffering from, or at risk of developing, a disease ordisorder that can be treated or prevented by administration of aneffective amount of CO. In general, the method comprises administeringto a patient a liquid composition containing CO dissolved in an amountsufficient for delivery of the CO to the patient, wherein administeringthe composition results in an effective treatment on a disease ordisorder that is treatable with CO. Unlike prior attempts to administerCO via direct inhalation of gaseous CO into the lungs, the presentinvention uses a liquid composition that is administered via the GItract or IV to deliver gaseous CO in vivo to the patient. In exemplaryembodiments, the liquid composition is administered orally by way ofdrinking of the composition. The method of the invention can bepracticed to treat a chronic disease or disorder, i.e., the step ofadministering can be repeated multiple times over a relatively longperiod of time (e.g., years) or over a relatively short period of time(e.g., a few days, a week, or the length of duration of an acute episodeof a disease or disorder). The method can be practiced as a therapeuticmethod to treat an active disease or disorder. Alternatively, the methodcan be practiced to prevent development of a disease or disorder. Themethod further can be practiced to reduce the likelihood of developing adisease or disorder or to reduce the frequency and/or severity ofclinical symptoms of a disease or disorder and/or its consequences onorgan and body function.

In yet another aspect of the invention, methods of making a liquidcomposition containing CO dissolved in a treatment-effective amount areprovided. In general, the method comprises dissolving CO in a liquidunder conditions that allow for relatively high amounts of CO todissolve into the liquid. Suitable conditions for aqueous compositionsinclude relatively high pressure, relatively low temperature, or acombination of both. Suitable conditions for lipid/oil basedcompositions might not require relatively high pressure or relativelylow temperature. In preferred embodiments, the method further comprisesdispensing the liquid into a container, prior to, during, or afterdissolving of the CO into the liquid. Preferably, the container issealed upon dissolving of the CO into the liquid. Preferably, the headspace of the container comprises CO. In exemplary embodiments, theliquid composition is a beverage, such as one provided in a sealablecontainer. In exemplary embodiments, the liquid composition comprises awater-based composition in which the CO is dissolved. In other exemplaryembodiments, the liquid composition comprises an oil- or fat-based foamin which CO bubbles are entrapped. Preferably, the method provides aliquid composition in which CO is in gaseous form in solution orentrapped in bubbles in a foam, and is not complexed with a metal or oneor more Hb molecules or complexes.

An additional general aspect of the invention relates to a method ofdetermining the appropriate dosing of the liquid composition isprovided. The method generally comprises using one or more of thefollowing to determine an appropriate dosing regimen: lung function,patient hemoglobin and red blood cell measurements, patient blood volumeand the concentration of CO in the liquid. Techniques for performingsuch assays are known in the art and can be practiced without undue orexcessive experimentation. The method can be practiced on a regularbasis to monitor and, if necessary, adjust the dosing regimen. Inembodiments, the method is a method of determining appropriate dosing ofthe liquid, where the method comprises titrating upwards the dosage ofthe liquid to determine optimal treatment. The method can include themeasurement of one or more of the following: CO exhalation, lungfunction, CO-hemoglobin, and pharmacokinetics.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to various exemplary embodiments ofthe invention. It is to be understood that the following discussion ofexemplary embodiments is not intended as a limitation on the invention,as broadly disclosed herein. Rather, the following discussion isprovided to give the reader a more detailed understanding of certainaspects and features of the invention.

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Further, where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lowerlimit, unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Eachsmaller range between any stated value or intervening value in a statedrange and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range,and each range where either, neither, or both limits are included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a protein” includes aplurality of such proteins (including multiple copies of the sameprotein and multiple different proteins) and reference to “the patient”includes reference to one or more patients, and so forth. Furthermore,the use of terms that can be described using equivalent terms includethe use of those equivalent terms. Thus, for example, the use of theterm “subject” is to be understood to include the terms “animal”,“human”, and other terms used in the art to indicate one who is subjectto a medical treatment. As another example, the use of the term“disease” is to be understood to include the term “disorder” (and viceversa) and all other terms used in the art to indicate an abnormal oraberrant medical condition.

The present invention provides a liquid composition containing COdissolved in an amount sufficient for delivery of the CO to a patient,wherein delivery to the patient results in an effective treatment for adisease or disorder that is treatable with CO. Due, at least in part, tothe method of making the composition (as disclosed herein), the liquidcomposition of the present invention has a gaseous CO concentration thatcannot be achieved using techniques known in the art for creatinggaseous CO-containing compositions. More specifically, the compositionof the present invention has a dissolved CO concentration of at least 30milligrams per liter (mg/l). The upper limit of dissolved CO is dictatedonly by physics and chemistry.

In exemplary embodiments relating to aqueous compositions, theconcentration of dissolved CO will generally not exceed 400 mg/l. Forexample, an effective concentration for convenient dosing of CO to treatSCC and SCD is between about 100 mg/l and 400 mg/l, althoughconcentrations between about 50 mg/l and about 100 mg/l are alsosuitable for patients amenable to intake of relatively large volumes ofliquid during treatment periods. Likewise, concentrations on the lowerend of these ranges can be suitable for SCC or SCD patients, whotypically have relatively low Hb levels, and thus do not require as higha dose of CO as patients having more normal levels of Hb. Although theskilled artisan will immediately understand that all particular valueswithin the range of about 30 mg/l and about 400 mg/l are specificallycontemplated by this application, the following concentration values,and the various ranges defined by the collection of specific values,provide convenient reference points for the practitioner to developcompositions according to the present invention: 30 mg/l, 35 mg/l, 40mg/l, 45 mg/l, 50 mg/l, 55 mg/l, 57 mg/l, 60 mg/l 65 mg/l, 70 mg/l, 75mg/l, 78 mg/l, 80 mg/l, 85 mg/l, 90 mg/l, 95 mg/l, 100 mg/l, 105 mg/l,110 mg/l, 115 mg/l, 120 mg/l, 125 mg/l, 130 mg/l, 135 mg/l, 140 mg/l,145 mg/l, 150 mg/l, 155 mg/l, 160 mg/l, 165 mg/l, 170 mg/l, 175 mg/l,180 mg/l, 190 mg/l, 200 mg/l, 210 mg/l, 225 mg/l, 250 mg/l, 275 mg/l,300 mg/l, 325 mg/l, 350 mg/l, and 375 mg/l. Aqueous compositionsaccording to the present invention can comprise any specificconcentration value between 30 mg/land 400 mg/l, can comprise anyspecific concentration range between 30 mg/land 400 mg/l, or can containa concentration of dissolved CO of at least any of the concentrationvalues between 30 mg/land 400 mg/l.

In exemplary embodiments relating to lipid/oil/fat-based compositions,the concentration of dissolved CO will generally not exceed 4,400 mg/l.For example, an effective concentration for convenient dosing of CO totreat SCC and SCD is between about 500 mg/l and 4,400 mg/l, such asbetween about 500 mg/l and 4,000 mg/l, although concentrations betweenabout 75 mg/l and about 500 mg/l are also suitable for patients amenableto intake of relatively large volumes of such liquids during treatmentperiods. Likewise, concentrations on the lower end of these ranges canbe suitable for SCC or SCD patients, who typically have relatively lowHb levels, and thus do not require as high a dose of CO as patientshaving more normal levels of Hb. Although the skilled artisan willimmediately understand that all particular values within the range ofabout 30 mg/l and about 4,400 mg/l are specifically contemplated by thisapplication, the following concentration values, and the various rangesdefined by the collection of specific values, provide convenientreference points for the practitioner to develop compositions accordingto the present invention: 75 mg/l, 100 mg/l, 125 mg/l, 150 mg/l, 175mg/l, 200 mg/l, 225 mg/l, 250 mg/l, 275 mg/l, 300 mg/l, 325 mg/l, 350mg/l, 375 mg/l, 400 mg/l, 425 mg/l, 450 mg/l, 475 mg/l, 500 mg/l, 525mg/l, 550 mg/l, 575 mg/l, 600 mg/l, 625 mg/l, 650 mg/l, 675 mg/l, 700mg/l, 725 mg/l, 750 mg/l, 775 mg/l, 800 mg/l, 825 mg/l, 850 mg/l, 875mg/l, 900 mg/l, 925 mg/l, 950 mg/l, 975 mg/l, 1000 mg/l, 1025 mg/l, 1050mg/l, 1075 mg/l, 1100 mg/l, 1125 mg/l, 1150 mg/l, 1175 mg/l, 1200 mg/l,1225 mg/l, 1250 mg/l, 1275 mg/l, 1300 mg/l, 1325 mg/l, 1350 mg/l, 1375mg/l, 1400 mg/l, 1425 mg/l, 1450 mg/l, 1475 mg/l, 1500 mg/l, 1525 mg/l,1550 mg/l, 1575 mg/l, 1600 mg/l, 1625 mg/l, 1650 mg/l, 1675 mg/l, 1700mg/l, 1725 mg/l, 1750 mg/l, 1775 mg/l, 1800 mg/l, 1825 mg/, 1850 mg/l,1875 mg/l, 1900 mg/l, 1925 mg/l, 1950 mg/l, 1975 mg/l, 2000 mg/l, 2050mg/l, 2100 mg/l, 2150 mg/l, 2200 mg/l, 2250 mg/l, 2300 mg/l, 2350 mg/l,2400 mg/l, 2450 mg/l, 2500 mg/l, 2550 mg/l, 2600 mg/l, 2650 mg/l, 2700mg/l, 2750 mg/l, 2800 mg/l, 2850 mg/l, 2900 mg/l, 2950 mg/l, 3000 mg/l,3100 mg/l, 3200 mg/l, 3300 mg/l, 3400 mg/l, 3500 mg/l, 3600 mg/l, 3700mg/l, 3800 mg/l, 3900 mg/l, 4000 mg/l, 4100 mg/l, 4200 mg/l, 4300 mg/l,and 4400 mg/l. Lipid/oil/fat compositions according to the presentinvention can comprise any specific concentration value between 30 mg/land 4400 mg/l, can comprise any specific concentration range between 30mg/l and 4400 mg/l, or can contain a concentration of dissolved CO of atleast any of the concentration values between 30 mg/l and 4400 mg/l.

The liquid composition of the present invention is not particularlylimited in its components, although exemplary embodiments disclosedherein have shown to be superior in the amount of CO that can bedissolved. While not so limited in all embodiments, in exemplaryembodiments, the liquid composition of the invention is a water-basedcomposition. It is to be understood that the term “water-basedcomposition” includes all compositions comprising water as a solvent,including, but not limited to: compositions in which water is the solesolvent; water-oil mixtures (e.g., water-in-oil and oil-in-wateremulsions); aqueous solutions, suspensions, colloids, and dispersions;water-alcohol mixtures; and combinations of these.

It has unexpectedly been found that aqueous compositions comprising oneor more “complex” components provides superior CO-dissolving capacity.“Complex” components, as used herein, are substances that are polymericin nature, biologic in nature, such as those derived from fatty acids,or otherwise comprise at least one bonding interaction site for CO.Interactions can be physical (e.g., hydrophobic, Van der Waals), orchemical (e.g., ionic or covalent). Examples of complex componentsinclude, but are not limited to: proteins, polypeptides, and peptides;polysaccharides; lipids, fats, and oils; and alcohols having two or morecarbon atoms. In some embodiments, lipid, protein, or both are presentin the compositions. In these embodiments, it is preferable that thecombined amount of protein and lipid be greater than 5% (w/v), and evenas high as 40% lipid and protein, or higher. In some formulations, thecomposition comprises greater than 5% lipid and greater than 5% protein.

The precise chemical structures of the complex components are notparticularly limited. Rather, it is sufficient that the complexcomponents function to assist in increasing the solubility of CO in thecomposition. However, to aid the practitioner in selecting appropriatecomplex components, the following is a non-limiting listing of types ofcomplex components: proteins and fats/oils/lipids/triglycerides ofanimal derivation, such as those in milk; proteins andfats/oils/lipids/triglycerides of plant derivation; mono-, di-, andpoly-saccharides; vitamins; natural and artificial sweeteners; andnatural and artificial flavorings. Any and all of the various moleculesthat are encompassed within these groups are included as part of thepresent invention. Those of skill in the art will immediately recognizesuch molecules without the need for an exhaustive listing herein.

In embodiments, the liquid composition of the invention takes the formof a beverage for oral consumption. Non-limiting examples of beveragesaccording to the invention are: bottled water, such as fruit- orberry-flavored waters; dietary/nutritional supplements, such as thoseformulated for infants and young children (e.g., baby formula, such asSimilac® (Abbott, Abbott Park, Ill.) and Enfamil® (Mead Johnson &Company, Glenview, Ill.)) or adults (e.g., Ensure® (Abbott, Abbott Park,Ill.), and Peptamen® and Nutren® (Nestle, Vevey, Switzerland)); liquiddairy or dairy-based products, such as milk, cream, yoghurt, or amilkshake; liquid soy or soy-based products, such as soy milk or soyyoghurt; liquid rice or rice-based products, such as rice drinks; sportsdrinks or dietary supplements, such as whey protein based drinks andGatorade® (Pepsico, Purchase, N.Y.); coffee-based drinks, such as thosesupplemented with dairy products; and sugar-containing or sugar-freesodas. As discussed in more detail below, certain liquid compositions ofthe invention are supersaturated with CO at room temperature andatmospheric pressure. As such, certain beverages can be effervescent asa result of release of a portion of the supersaturated CO. Thiseffervescent property can enhance the patient's experience wheningesting the beverage, and can improve compliance with a dosingregimen.

In embodiments, the liquid composition of the invention takes the formof a foam- or gel-based food product. For example, in some embodiments,the liquid composition is provided in the form of a gel, such as agelatin or pudding, such as those commercially available under theJell-O® brand (KraftFoods, Inc., Glenview, Ill.). Yet again, in someembodiments, the liquid composition is provided in the form of a foam,such as those commercially available under the Coolwhip® brand(KraftFoods, Inc., Glenview, Ill.) and ReddiWip® brand (ConAgra Foods,Inc., Omaha, Nebr.).

A beverage or food product according to the invention can be provided ina container. In preferred embodiments, the container is a sealablecontainer of the type widely used for providing commercial beverages andfood products to the public. Non-limiting examples of sealablecontainers for holding the beverage and/or food product are: plasticbottles with twist on/off tops; aluminum cans with pop tops; glassbottles with twist on/off tops; and glass bottles with crimp-sealedaluminum tops or tops made of other pliable metals. For convenience indelivering an effective amount of CO to a subject in need, in preferredembodiments, the amount of beverage or food product in a singlecontainer is an adequate volume of beverage or food product to supply asingle dose of CO (e.g., 5 ml, 10 ml, 30 ml, 50 ml, 75 ml, 100 ml, 150ml, 177 ml, 180 ml, 237 ml., 300 ml, 355 ml, 500 ml, one liter). Assuch, it is recognized that the liquid compositions of the invention canbe provided such that a daily dosage for treatment of a disease ordisorder (e.g., a symptom thereof) is conveniently provided in volumesof about 3 liters or less, such as 2.5 liters, 2 liters, 1.8 liters, 1.5liters, 1 liter, 330 ml, 300 nil, 180 nil, 30 ml, or less. However, itis to be understood that, in situations where the beverage or foodproduct is supplied in a re-sealable container (e.g., a bottle with atwist on/off cap), the amount of beverage or food product in thecontainer can represent more than one dose of CO.

The invention thus provides products for practicing the method of theinvention (discussed in detail below). The products can be productstypically associated with medical procedures, such as solutions andcontainers containing solutions (e.g., IV bags). Alternatively, theproducts can be in the form of more easily administered products, suchas canned or bottled solutions or foods. The technology is simple toapply, unlikely to be associated with significant side effects, andacceptable to the affected population, thus resulting in reliableutilization of the treatment method.

In summary, in various exemplary embodiments, the present inventionprovides a liquid composition comprising dissolved gaseous carbonmonoxide (CO) in an amount of from 30 mg/l to 4400 mg/l in the liquidcomposition. In embodiments, the composition comprises dissolved gaseousCO in an amount of from 50 mg/l to 400 mg/l, from 75 mg/l to 750 mg/l,and from 550 mg/l to 4400 mg/l. In general, the dissolved CO is presentin an amount sufficient to prevent or treat at least one clinicalsymptom of a disease or disorder affected by CO. Various diseases anddisorders treatable with the composition are discussed below. Inembodiments, in addition to dissolved gaseous CO, the compositionfurther comprises at least one of: protein, lipid, fat, triglyceride,complex carbohydrate, sugar, sugar substitute, fruit juice,carbohydrate, cellulose, fiber, citric acid, artificial flavoring,natural flavoring, gum, pectin, ascorbic acid, preservative, saponin,oil, oil emulsion, pH buffer, and a salt. For the liquid portion of thecomposition, in exemplary embodiments, water, ethanol, or both are used.In embodiments, the liquid composition is one in which the amount ofdissolved CO is greater than occurs under ambient temperature andpressure and at a pH of 7.0 and/or at pH of 7.0, atmospheric pressure,and 21° C. For example, the dissolved CO can be two or more times theamount dissolved under ambient temperature and pressure and at a pH of7.0 and/or at pH of 7.0, atmospheric pressure, and 21° C.

As mentioned above, the liquid composition can comprise dissolvedgaseous CO, or gaseous CO entrapped in bubbles, in an amount of at least0.03 grams of gas per kilogram of water or other liquid (i.e., at least30 mg/l). In embodiments, the amount of dissolved or entrapped CO isgreater than 0.04 grams of gas per kilogram of water or other liquid. Inembodiments, the amount of dissolved CO is equal to or greater than theamount that occurs under two atmospheres of pressure at 10° C. and at apH of 7.0. In certain embodiments, the liquid composition is one inwhich dissolved or entrapped CO can be administered orally,intravenously, or otherwise through the gastrointestinal tract, and thatcontains no potentially toxic constituents other than CO.

The liquid composition of the invention enables one to treat a patientsuffering from a disease or disorder, which is amenable to treatmentwith CO. Thus, one aspect of the present invention is a method fortreating a patient suffering from, or at risk of developing, a diseaseor disorder and/or suffering from consequences of the disease ordisorder on organ function that can be treated or prevented byadministration of an effective amount of CO. Thought of another way, themethod can be considered a method of treating a disease or disorder thatcan be treated or prevented by administration of an effective amount ofCO. Broadly speaking, the method comprises administering to a patient aliquid composition comprising a therapeutically-effective or aprophylactically-effective amount of dissolved gaseous CO, whereinadministering the composition results in an effective treatment for adisease or disorder that is treatable with CO. Administering can beaccomplished by any suitable technique known in the art. However, it hasbeen determined that administering via the GI tract or via IV infusionor injection is superior to other routes of administration.Administering via the GI tract is preferably accomplished through oralingestion (e.g., drinking). However, the invention contemplatesadministration via other points in the GI tract, such as by way of anenema or by way of direct delivery to the small or large intestine. Theinvention also contemplates delivery via inhalation of an aerosolized ornebulized liquid and delivery via IP, IM, organ catheter, andsubcutaneous injection. However, for reasons discussed herein, theseroutes of administration are less preferred.

Many diseases and disorders that can be treated with CO are chronic innature. As such, long-term treatment is often necessary. The presentinvention is particularly well suited for treatment of such chronicdiseases and disorders because it allows for long-term delivery ofpre-defined and precisely regulated amounts of gaseous CO to a patient.Unlike delivery of airborne CO to the lungs via inhalation, the presentinvention, by delivery of gaseous CO dissolved in a liquid to the GItract or via IV infusion to the blood system, delivers CO through anorgan other than the lungs. While adjustments in the dosing regimen willlikely be needed over a long course of treatment, avoidance of the lungsas the site of administration avoids a complicating factor for preciseand accurate dosing, which is a significant consideration in view of thesmall difference in concentration of CO between therapeutic levels andtoxic levels. The same can be said for administration via IV infusion,although oral ingestion is much more convenient and cost-effective.

The present invention is also well suited for treatment of acuteepisodes of diseases and disorders that can be treated with CO. Deliveryof a composition according to the invention to the GI tract or to theblood stream allows for rapid dosing of CO to a patient. Systemictherapeutic CO levels can be achieved rapidly, thus allowing forreduction in clinical symptoms of a disease or disorder. Among thebenefits provided by the invention is the limiting of the extent ofdamaged tissue and thus the extent of pain associated with SCC. Morespecifically, by quickly raising the CO content of the blood, the damagecaused during SCC can be limited to the site of the original pain, thusreducing spread of the damage and pain. Furthermore, because a patientexperiencing SCC will have been diagnosed with SCD (except, potentially,for the first episode of SCC), the appropriate amount of CO to bedelivered can easily be determined, thus allowing for accurate andprecise delivery of an effective, but not toxic, amount of CO to thepatient.

The method of treating according to the invention is a method thattreats patients suffering from diseases and disorders that can betreated with CO. Because the effective bioactive agent is known, and itsrole in treating the disease or disorder is known, the method accordingto the invention can be practiced both therapeutically to treat adisease or disorder, and prophylactically to prevent or delay onset ofthe clinical symptoms of a disease or disorder. That is, because CO isthe active agent that reduces and, ultimately, eliminates the clinicalsymptoms of a disease or disorder encompassed by the present invention,it also is the active agent that prevents or delays onset of a clinicalsymptom of a disease or disorder encompassed by the present invention.For example, in the case of SCD, prophylactic treatment with CO canreduce or eliminate formation of sickle-shaped red blood cells, thuspreventing or delaying onset of SCC and mitigating the complications ofSCC. Of course, those of skill in the art will understand that the term“prevent” does not imply absolute prevention of any development andprogression of a disease or disorder, but instead indicates blocking, toat least some extent, of the natural development or progression of thedisease or disorder.

The method of the invention comprises administering a CO-containingliquid composition to a patient in need thereof. Although the inventioncontemplates performing the administering step a single time, the stepof administering can be repeated any number of times. Indeed, inpreferred embodiments, the step of administering is repeated asufficient number of times to achieve a carbonmonoxy-hemoglobin (hereinreferred to as “CO-Hb”) concentration suitable for the disease ordisorder being treated. For example, for treatment of SCD, an averageCO-Hb concentration of between 3% and 15%, more preferably between 3%and 12%, most preferably between 3% and 9%, such as about 3% to 6%, isdesirable. It is known that the half-life of the alpha-phase of CO inthe human bloodstream is about 4-6 hours. Therefore, an average CO-Hbconcentration in the bloodstream can be achieved, for example, through adosing regimen of four or fewer doses per day, preferably equallyspaced, such as four doses per day (i.e., every six hours), three dosesper day (i.e., one dose every eight hours), two doses per day (i.e.,every twelve hours), or one dose per day. Due to the relatively high COconcentration achievable in a liquid composition according to thepresent invention, relatively small volumes of liquid composition can beadministered per dose or per day, such as: 2 liters per day, 1.5 literper day, 1 liter per day, 0.7 liter per day, 0.5 liter per day, 0.25liter per day, and 0.1 liter per day, and other amounts disclosedherein. Dosing of small volumes improves patient compliance and providesan overall superior outcome for the patient. As there is no knowndetrimental effect to long-term exposure to CO at these levels, thedaily administration can be performed indefinitely. Those of skill inthe art will recognize that this concept is equally applicable to otherdiseases and disorders treatable with CO, and that the average CO-Hblevel suitable for treatment of those diseases and disorders might bedifferent, but can easily be determined.

The present invention is based, at least in part, on the understandingthat dosing with CO must be well controlled due to the fact that thetoxic level of CO is close to the therapeutic level. More specifically,it is generally recognized that levels of CO in the bloodstream thatresult in up to about 15% CO-Hb show effectiveness at reducing SCCwithout significant deleterious side effects or toxicity. However, it isalso generally recognized that, at CO-Hb levels between about 16% and20%, some toxicity is seen, and that at CO-Hb levels over 20%, toxicityand side effects are routinely seen. The present invention takes intoaccount the closeness of effective dose levels without substantialuntoward side effects or toxicity and dose levels that result intoxicity and side effects. The present invention provides products andtreatment methods that conveniently, precisely, and reproducibly achievean effective dosing of CO to patients while avoiding toxic levels and/orside effects.

Among the diseases and disorders that are encompassed by this invention,mention can be made of: hematological diseases, inflammatory diseases,ischemic diseases, and cardiovascular diseases. For example, thetechnology can be useful in treatment of hematological diseases suchSCD. Further, the technology can be useful in inflammatory diseases,such as inflammatory bowel disease, Crohn's disease, ulcerative colitis,multiple sclerosis, rheumatoid arthritis, coronary ischemia, systemicinflammation, and inflammation associated with transplantation. Yetagain, the technology can be useful in treatment of ischemic disease,such as, coronary ischemia, neural ischemia, organ ischemia, andshock-induced ischemia. Likewise, the technology can be useful intreating cardiovascular diseases, such as: vascular disease,ischemia/reperfusion, and decrease in lung function. Other non-limitingdiseases and disorders that are applicable to the present inventioninclude gastrointestinal diseases, such as post-operative ileus andtransplantation; sepsis; kidney disease; and liver disease, such asischemia/reperfusion and burn injury.

Exemplary embodiments of the invention relate to diseases of the bloodor hematological diseases. Among the hematological diseases that can betreated according to the present invention are those involving abnormalhemoglobin, such as SCD, Hemoglobin C Disease, Hemoglobin SC Disease,and Hemoglobin S beta-thalassemia.

With regard to SCD, the present invention provides a new way to treatthe disease, including prevention and treatment of SCC using gaseous CO.In treatment of SCC, the CO is delivered to a subject in need (i.e.,patient) in an amount that affects at least one clinical symptom of SCC.In preferred embodiments, the method of treatment reduces or eliminatesat least one clinical symptom of SCC, but in an amount that does notinduce unacceptable levels of toxicity or adverse side-effects. Inprevention of SCC, the CO is delivered to a subject in need (i.e.,patient) on a regular basis in an amount that affects the number of SCCexperienced by the subject in need. In preferred embodiments, the methodof prevention reduces the number or severity of SCC experienced by thesubject in need. Administration for treatment of SCD and SCC can bethrough any route suitable for a liquid. In preferred embodiments,administration is through oral ingestion of the liquid or via injectionor infusion into the bloodstream. Although CO is toxic in highconcentrations, it is non-toxic at the relatively low concentrationsuseful according to the present invention. Furthermore, because CO bindsto hemoglobin S (“Hb-S”) at a much higher affinity than does oxygen, andbecause Hb must be in non-gas bound configuration to polymerize (whichresults in sickle cell development), relatively low doses of CO can beused to prevent and treat SCD and SCC. The method may also compriseadministering CO to a subject on a regular basis in an amount that issufficient to reduce the number of SCC experienced by the patient and/orreduce the resulting organ damage caused by sickling. Organs affectedinclude, but are not limited to, lungs, brain, heart, kidneys, bone,spleen, liver, endocrine glands, and male sex organs. The impact ofsickling over time extends the degree of organ damage. In embodiments,the method is repeated regularly to provide a chronic treatment regimen.

While there are a number of ways to administer the CO to treat SCD andSCC, the most convenient is by way of or administration of a CO-infusedliquid. CO absorbed through the GI system or injected into thebloodstream rapidly attaches to Hb, thus providing an effective andrapid treatment.

Treatment of SCC according to the methods of the invention can have thefollowing effects for acute use: it can shorten the crises and alleviatethe severity of the crises. Treatment of SCC according to the methods ofthe invention can have the following effects for chronic use: it candecrease the frequency of crises, it can decrease the severity of thecrises, it can decrease the extent, progression, and frequency of organdamage, and it can outright prevent crises.

Delivery of CO to a patient via administration of liquid is mostconveniently achieved using generally available containers, such as ametal can or glass or plastic bottle. The liquid and type of containeris not particularly limited, with the exception that the liquid mustallow for adequate levels of CO to dissolve, while the container must befabricated of material that is sufficiently impermeable to CO andsufficiently pressure resistant. Of course, the practitioner will needto adjust the composition of the liquid in some situations to optimizeCO dissolving into the liquid and thus absorption into the body of thepatient. For example, the salinity, pH, sugar content, amount of organiccompounds (e.g., alcohol), protein content, lipid content, etc. can bevaried to optimize the taste, consistency, etc. of the composition, andthe amount of CO that dissolves into the liquid and also to optimize COabsorption once delivered to the patient. Also, the pressure,temperature, and components in the composition during CO dissolution canbe varied to optimize the amount of CO that dissolves into the liquid.Likewise, non-aqueous solutions of various constituents can be used, aswell as aqueous or non-aqueous compositions that include undissolvedconstituents.

Among the many containers that can be used, mention may be made of:glass bottles, plastic bottles; and aluminum cans, and containersfabricated from combinations of glass, plastic, and aluminum and othermetals.

CO has a number of medical properties and has shown promise in treatinga variety of diseases. The primary challenge in using CO as atherapeutic given its potential toxicity is to deliver a small butsufficient amount of CO to treat disease without causing harm. Untilnow, it has not proven possible to do this.

There are a number of significant limitations to using the delivery ofCO dissolved in liquid and used through the GI tract or IV fortherapeutic or prophylactic purposes. These limitations have beenovercome in the present invention. The primary barrier to date has beendelivering sufficient quantities of CO. The solubility of CO in aqueoussolutions is low at room temperature and normal atmospheric pressure(ATM). Thus, using approaches previously proposed in the art, which relyon dissolving CO into an aqueous composition at room temperature and atatmospheric pressure, the volume of liquid necessary to deliver atherapeutic dose of CO is logistically prohibitive. However, using thepresent invention, therapeutically useful levels of CO can be achieved.Table 1 provides a summary of volumes necessary to achieve a therapeuticdose providing an average increase of 9% CO-Hb, which provides anaverage CO-Hb level in the blood of about 7% on average without risingabove 15% at any given time. The values are presented for SCD patients.Table 2 provides a similar summary of volumes necessary to treatpatients having normal levels of Hb and normal lung function.

TABLE 1 CO Concentration Volume of Liquid Necessary Daily Achievable inNeeded to Achieve Volume of Liquid to Aqueous Increase of 9% of AchieveAverage Temperature, Composition CO-Hb in SCD Increase of 9% Pressure(mg/L water) Patients CO-Hb 21° C.; 1 ATM 25 2.54 L per 12 hrs 5.1 L  2°C.; 1 ATM 40 1.59 L per 12 hrs 3.2 L  2° C.; 2 ATM 80 0.79 L per 12 hrs1.6 L  2° C.; 3 ATM 120 0.53 L per 12 hrs 1.1 L  2° C.; 5 ATM 200 0.32 Lper 12 hrs 0.6 L  2° C.; 10 ATM 400 0.15 L per 12 hrs 0.3 L

TABLE 2 CO Volume of Liquid Necessary Daily Concentration Needed toAchieve Volume of Achievable in Increase of 9% of Liquid to AchieveAqueous CO-Hb in Average Temperature, Composition “Normal” Increase of9% Pressure (mg/L water) Patients CO-Hb 21° C.; 1 ATM 25 4.52 L per 8hrs 13.6 L   2° C.; 1 ATM 40 2.83 L per 8 hrs 8.5 L  2° C.; 2 ATM 801.41 L per 8 hrs 4.2 L  2° C.; 3 ATM 120 0.94 L per 8 hrs 2.8 L  2° C.;5 ATM 200 0.57 L per 8 hrs 1.7 L  2° C.; 10 ATM 400 0.28 L per 8 hrs 0.8L

Tables 1 and 2 demonstrate that under ambient pressure, even at lowtemperature, it is not possible to achieve a CO concentration insolution that enables a therapeutic dose of CO using a volume of aqueoussolution that can reasonably be taken orally by a patient in a 24 hourperiod.

In order to achieve a higher CO concentration that allows a volume ofaqueous solution that can reasonably be taken orally in a 24 hourperiod, in the present invention a number of improvements to priorattempts in the art have been made.

First, in the present invention, in particular as it relates toembodiments wherein aqueous compositions are involved, CO is dissolvedin an aqueous liquid under higher than ambient pressure. The use ofpressure to dissolve the CO increases the achievable concentration of COin solution. Tables 1 and 2 demonstrate the significantly increased COconcentrations achievable under pressure and the correspondingsubstantial decrease in the necessary amount of aqueous solution neededto provide an effective dose of CO. One of skill in the art willimmediately recognize that lowering of the temperature below 2° C. andincreasing the pressure above 10 ATM can achieve an even greaterconcentration of CO dissolved in the aqueous solution, and that thevalues presented in Table 1 are for demonstration purposes only.Although raising the pressure above 10 ATM might not be commerciallypracticable, it is technologically feasible at this time.

Tables 1 and 2 demonstrate that under greater than ambient pressure andlow temperature, it is possible to achieve a CO concentration insolution that enables a therapeutic dose of CO in a volume of aqueoussolution that can reasonably be taken orally by a patient in a 24 hourperiod. The volumes needed to be administered per day are well withinamounts taken daily by many people. Thus, compliance by patients shouldbe vastly improved as compared to any attempts at treatment withCO-containing liquids known in the art.

Second, the use of a GI formulation (e.g., a formulation for oraladministration) overcomes a challenge of using a CO-infused aqueouscomposition as a therapeutic. An aqueous oral or GI formulation allowsthe use of a solution with CO dissolved under higher than ambientpressure and low temperature, enabling a therapeutic dose of CO. When COis dissolved into solution under greater than ambient pressure, and thesolution is then moved into an ambient pressure environment upondelivery of the solution, the CO gas will bubble out of solution(similar to liquid solutions containing dissolved CO₂). Bubbles of gaspose a substantial danger to a patient in many delivery mechanisms, suchas IP and intravenous delivery, amongst others. However, the bubbling ofCO gas out of solution in the stomach or intestine does not pose asubstantial safety risk. Thus, the use of a delivery route that allowsthe bubbling of CO out of solution enables the use of this invention intherapeutic indications that require a high CO concentration. Also,dissolving CO into solution under temperatures close to or below 0° C.increases the CO saturation of the solution, as compared to dissolvingCO at room temperature. The administration of a very cold solution posesdiscomfort and danger to a patient in many delivery mechanisms, such asIP and intravenous delivery, amongst others. However, the use of a coldliquid in the stomach or intestine does not pose a substantial safetyrisk or substantial discomfort. Thus, the use of a delivery route thatallows the administration of a cold solution enables the use of thisinvention in therapeutic indications that require a high COconcentration. Furthermore, while the present invention contemplatesdissolving CO into a liquid at relatively low temperatures, the CO neednot be delivered to a patient by way of a cold liquid. For example, aCO-containing liquid composition can be prepared in a sealablecontainer. Once sealed, the container need not be maintained at a coldtemperature for the CO to stay in solution. Although a greater amount ofCO will escape from solution if the container is opened at highertemperatures than at lower temperatures, ingestion (or other delivery)of the liquid to the patient in a relatively short time period afteropening of the container will minimize loss of CO from the liquid.

Third, the present invention recognizes that complex liquid compositionsare superior to water or relatively simple aqueous compositions fortherapeutic delivery of CO. Prior attempts at delivering CO to cellsused CO dissolved in water or in aqueous solutions of water and salts.According to preferred embodiments of the present invention, the liquidcomposition for delivery of CO is an aqueous composition that containsone or more relatively complex molecules, such as proteins, lipids,oils, alcohols, and/or carbohydrates. It has been found that thepresence of these complex molecules allows greater CO solubility ascompared to compositions lacking them. As such, inclusion of thesecomplex molecules overcomes a challenge of using CO as a therapeutic.Preferred embodiments of the invention thus include administration ofcomplex aqueous compositions comprising therapeutic concentrations of COmixed with one or more lipids, proteins, or other substances that aid inincreasing the concentration of CO in the aqueous composition.

Fourth, another characteristic of the present invention that overcomes achallenge with using CO as a therapeutic is the use in patients with lowHb concentrations, including those patients with SCD. As mentionedabove, the amount of liquid that can be administered to a patient is alimiting factor given the relatively low solubility of CO in liquids.However, because the amount of Hb is lower in certain patientpopulations, such as those with SCD, therapeutic dosage levels of CO arelikewise lower in such patients. The high concentration of CO possiblein liquids according to the present invention allows greater ease ofuse, improved patient compliance, and an overall improvement intherapeutic effect, as compared to other treatments proposed in the art.

As discussed above, the invention provides a method of treating apatient suffering from a disease or disorder, or at risk of developing adisease or disorder, that can be treated or prevented by administrationof CO. The invention thus provides for the use of a liquid compositioncomprising a therapeutically effective amount of CO in the treatment ofa disease or disorder, or for the prevention of a disease or disorder,that can be treated or prevented by administration of CO. The inventionfurther provides for the use of a liquid composition comprising atherapeutically effective amount of CO in the preparation of a medicinalcomposition for the treatment or prevention of a disease or disorderthat can be treated with CO.

In yet another general aspect of the invention, methods of making aliquid composition containing CO dissolved in a treatment-effectiveamount are provided. Previous attempts in the art to create a liquidcomposition containing dissolved gaseous CO involved dissolving CO in anaqueous liquid at ambient temperature and atmospheric pressure,resulting in a composition that is unsuitable for use as an in vivotherapeutic or prophylactic agent due to the low CO content in theliquid. The present invention, in contrast, achieves a therapeutically-and prophylactically-effective agent through the use of a preparationmethod that includes the use of high pressure, cold temperature, or acombination of the two. Preferably, the method also includes the use ofa liquid composition that includes one or more complex components, whichaid in increasing the concentration of CO dissolved in the composition.

The method of making a CO-containing liquid composition typicallycomprises subjecting a liquid composition to a high pressure whileexposing the composition to gaseous CO for a sufficient amount of timeto achieve an adequately high concentration of CO in the liquidcomposition to provide a therapeutically-effective and/orprophylactically-effective composition. However, it is to be noted that,in certain embodiments in which the compositions comprise lipids, fats,or oils, introduction of therapeutic levels of CO might not require theuse of higher than atmospheric pressure. In preferred embodiments, thestep of exposing comprises infusing CO into the liquid composition bybubbling through a cannula, aerator, or other equivalent device ormethod. In some embodiments, the liquid composition is subjected tomixing or stirring during the process of exposing to CO in order tofacilitate dissolving of the CO into the liquid composition.

While not required, in embodiments relating to commercial production ofthe liquid composition, it is preferred that the CO-containing liquidcomposition be sealed in a CO-impermeable container to preclude loss ofdissolved CO over time. Accordingly, in embodiments, the method ofmaking a CO-containing liquid composition comprises dispensing theliquid composition into a sealable CO-impermeable container and sealingthe container after an appropriate amount of CO has been dissolved inthe liquid composition. Dispensing of the liquid can be performedbefore, during, or after dissolving CO into the liquid composition.Typically, sealing will be performed under CO gas and under greater thanatmospheric conditions to minimize loss of dissolved CO during theprocess of sealing. As such, it is preferable that the container andsealing mechanism (e.g., cap, top) are resistant to the pressures usedduring dissolving of CO into the composition (e.g., from about 1.1 ATMto about 8 ATM or higher). As discussed above, any number of sealablecontainers and caps are known in the art of bottling and canning ofliquids, and any of these can be used within the context of the presentinvention.

The method of making a CO-containing liquid composition relies on theuse of greater than atmospheric pressure (i.e., “high” pressure) toincrease the amount of CO dissolved in the liquid composition. Accordingto the invention, at least 1.1 atmospheres (ATM) of pressure is used inthe process of introducing CO into the liquid composition. Preferably,where high pressure is used, at least 1.2 ATM is used. In certainexemplary embodiments, 2 ATM, 3 ATM, or 5 ATM is used duringintroduction of CO into the liquid composition. It is contemplated bythe invention that pressures above 5 ATM are also suitable, such as 6ATM, 7 ATM, 8 ATM, and 10 ATM or higher. In accordance with thediscussion concerning ranges set forth above, the skilled artisan willrecognize that all specific values, and all possible ranges, fallingwithin the atmospheric conditions discussed herein are contemplated aspart of the invention, and that there is no need to specifically reciteeach and every possible value and range in order for the skilled artisanto recognize that all such values and ranges are envisioned as part ofthe invention. Furthermore, it is to be understood that variations inthe source of CO, the equipment used to introduce the CO into the liquidcomposition, the pressure of the CO being used (i.e., volume suppliedper unit time), and the atmospheric pressures used will affect theamount of time required to achieve a suitable concentration of CO in thecomposition. Those of skill in the art can easily identify the correctparameters to achieve a CO-containing composition using only standard,straightforward procedures known in the art, without any undue orexcessive experimentation.

The method of making a CO-containing liquid composition preferablycomprises subjecting the composition to a low temperature during thestep of exposing the liquid to CO. It has been found that lowering thetemperature from ambient room temperature (about 21° C.) to about 2° C.increases the amount of CO that is dissolved in the liquid composition.According to the invention, a low temperature is a temperature at orbelow 4° C., preferably at or below 2° C., such as 1° C., 0° C., −1° C.,−2° C., −3° C., −4° C., −5° C., −6° C., −10° C., −12° C., −14° C., −16°C., −18°, −20° C., or below. In embodiments where the composition isexposed to a low temperature, it is preferred that the CO-containingliquid composition be sealed to preclude loss of dissolved CO over time.Accordingly, in embodiments, the method of making a CO-containing liquidcomposition comprises dispensing the liquid composition into a sealablecontainer and sealing the container after an appropriate amount of COhas been dissolved in the liquid composition. Dispensing of the liquidcan be performed before, during, or after dissolving CO into the liquidcomposition. Typically, sealing will be performed under low temperatureto minimize loss of dissolved CO during the process of sealing. Asdiscussed above, any number of sealable containers and caps are known inthe art of bottling and canning of liquids, and any of these can be usedwithin the context of the present invention. For the sake of clarity, inembodiments where both high pressure and low temperature are used, it ispreferred that sealing be performed under both high pressure and lowtemperature, although the same pressure and temperature used fordissolving CO into the liquid need not be used. Further, it is to beunderstood that, once the CO-containing liquid is sealed in a containerunder CO gas, it is not necessary to maintain the sealed container athigh pressure and low temperature, as the sealed container will notallow dissolved CO to escape from solution.

The present invention identifies three important parameters forachieving a liquid composition having a therapeutically- and/orprophylactically-effective amount of dissolved gaseous CO: introducinggaseous CO under high pressure; introducing gaseous CO under lowtemperature; and the presence of complex substances in the liquidcomposition. However, it is to be understood that other parameters canbe adjusted to improve or otherwise alter the concentration of CO insolution, or simply to alter the overall taste and consistency. Theseparameters can be adjusted for any number of reasons, including, but notlimited to: altering the taste of the liquid; altering the sweetness,tartness, or tang of the liquid; altering the pH of the liquid; alteringthe salinity of the liquid; and altering the consistency of the liquid.It has been found that alterations in many parameters do notsignificantly affect the overall CO carrying capacity of a liquidcomposition, with the main exceptions of alteration of pressure,temperature, and presence of complex components, as described above. Forexample, tests show that, for a given pressure, temperature, and complexcomponent combination, variations in pH, simple sugar concentrations,and salt concentrations have little effect on dissolved CO levels.Accordingly, the practitioner may adjust various parameters to suit aparticular need without departing from the concept of the invention.

EXAMPLES

The invention will be further explained by the following Examples, whichare intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way.

Example 1 Exemplary Formulations

The present invention provides an advancement over the prior art byproviding a liquid composition comprising therapeutically- and/orprophylactically-effective amounts of dissolved gaseous CO in dosageforms. The formulations are based on three main criteria: the level ofgas-phase pressure during dissolving of CO into the liquid composition;the temperature during dissolving of CO into the liquid composition; andthe presence of complex components in the liquid composition. Tables 3-5show data indicating the role of proteins, fats, and other complexcomponents in raising the dissolved CO concentration in liquidcompositions according to the present invention.

Referring to Table 3, it can be seen that CO can be dissolved in liquidcompositions comprising fats and proteins (Ensure® (Abbott, Abbott Park,Ill.), 10%-50% in water) and standard cream from cow milk (50% cream inwater, v/v) at concentrations higher than achievable in water alone, onthe order of at least 28 mg/l. Although improved dissolved COconcentrations can be achieved at 1 ATM and 21° C. using low levels ofproteins and fats (e.g., 10% Ensure®), the amount of dissolved CO can beincreased by increasing the amounts of proteins and fats, and byincreasing the gas-phase pressure applied during the dissolving process.

TABLE 3 CO Concentrations with Various Protein and Fat ContainingSolutions (~1 atmosphere pressure; Solutions in Water) CO ConcentrationSolution Temperature (mg/L) N 10% Ensure ® −21° C. 28.1 3 20% Ensure ®−21° C. 34.2 5 30% Ensure ® −21° C. 43.2 4 40% Ensure ® −21° C. 46.3 250% Ensure ® −21° C. 59.8 2 50% Cream −21° C. 63.9 10 50% Cream  −2° C.74.9 10

Looking now at Table 4, one can see that the amount of pressure providedduring the process of dissolving CO into a water-containing liquidcomposition is an important factor in achieving a composition withtherapeutic levels of CO. More specifically, Table 4 shows that, for agiven percent of Ensure® or standard cream, increasing the pressureduring dissolving of CO results in a significant increase in the amountof CO infused into the composition.

TABLE 4 CO Concentrations with Protein and Fat Containing Solutions atVarious Pressures (Solutions in Water) CO Concentration Solution Temp.Pressure (mg/L) 50% Ensure ® −21° C. 1 ATM 60 50% Ensure ® −21° C. 2 ATM120 50% Ensure ® −21° C. 3 ATM 180 50% Ensure ® −21° C. 5 ATM 300 50%Ensure ® −21° C. 10 ATM  600 50% Cream  −2° C. 1 ATM 75 50% Cream  −2°C. 2 ATM 150 50% Cream  −2° C. 3 ATM 225 50% Cream  −2° C. 5 ATM 375 50%Cream  −2° C. 10 ATM  750

Table 5 shows a comparison of the amounts of liquid composition neededto provide a therapeutic dose of CO to non-SCD subjects (referred to as“normal” patients) using aqueous compositions and compositionscomprising proteins and fats (i.e., a 50% cream composition). As can beseen from the Table, the volume of composition needed to be administeredis one-half or less for liquid compositions comprising proteins and fatsas compared to simple aqueous compositions.

TABLE 5 Volumes Necessary to Achieve a Therapeutic Dose of CO in NormalPatients (Therapeutic dose assumed at an average increase of 5% CO-Hb)Necessary Daily Volume of Non- CO Necessary Daily CO Aqueous SolutionConcentration Volume of Aqueous Concentration (50% Cream) to in AqueousSolution to Achieve in Non-aqueous Achieve Average Temperature, SolutionAverage Increase of Solution (mg/L Increase of 5% Pressure (mg/L water)5% CO-Hb water) CO-Hb 2° C.; 1 ATM 40 8.4 L 75 3.6 L 2° C.; 2 ATM 80 4.2L 150 1.8 L 2° C.; 3 ATM 120 2.8 L 225 1.2 L 2° C.; 5 ATM 200 1.7 L 3750.7 L 2° C.; 10 ATM 400 0.8 L 750 0.4 L

As can be seen from Tables 3-5, compositions containing therapeutically-and prophylactically-effective levels of CO can be produced according tothe present invention. Further, the methods according to the invention,and the compositions prepared using the methods, are superior incapturing CO in solution through the use of higher than 1 ATM ofpressure, temperatures lower than 21° C., and compositions comprisingcomplex substances, such as proteins and/or fats.

Example 2 In Vivo Efficacy of Compositions of the Invention

The ability of compositions according to the present invention todeliver therapeutic levels of CO to the bloodstream of humans and ratshas been established, as reported in this Example. More specifically, anaqueous formulation was prepared by dissolving CO into water atapproximately 2° C. and approximately 1.2 ATM, resulting in acomposition containing a concentration of CO of 0.05 g/L. Approximately350 ml of the CO-infused water was taken orally twice at an interval of1.5 hours by the normal volunteer, with the following results (Table 6).These results align to the expected stoichiometric results withinexpected error bands and loss of CO over time:

TABLE 6 Concentration of CO-Hb in blood of normal subject after dosingConcentration Time (hours) (% CO-Hb) 0 (baseline) 1.7 1.5 2.8 3.0 3.4This experiment shows not only that therapeutically-effective levels ofCO can be infused into liquid compositions according to the invention,but that such compositions can be orally ingested and can delivertherapeutic doses of CO to the bloodstream, as assayed by CO-Hbconcentrations.

To further show the effectiveness of the present invention in creatingtherapeutically-effective liquid compositions for delivery of CO, aliquid composition comprising protein and fat was prepared according tothe process of the invention, and was tested for its CO-deliveringactivity in rats. More specifically, a formulation of 50% cream in waterwas prepared and CO was introduced into it at approximately 2° C. andapproximately 1.2 ATM, resulting in a composition having a concentrationof 0.075 g/L (75 mg/l). Three (3) ml. of the formulation wasadministered to rats by way of gavage, and the resulting CO-Hb levelswere assayed and compared to a negative control that received a non-COcontaining liquid via gavage.

More specifically, laboratory rats weighing 250-300 grams were treatedby gavage with either: 1) no gavage (negative control); 2) 3 ml ofCO-infused water twice at times 0 and 1 hour; and 3) 3m1 of CO-infused50% cream twice at times 0 and about 1.25 hours. Blood sampling wascarried out at approximately 1.5 hours after the second gavage for eachrat.

The results of the tests are presented in Table 7. The results show thatCO can be delivered to the rat bloodstream in an amount that iseffective to treat diseases and disorders associated with CO-effectiveoutcomes via an oral route using compositions according to the presentinvention. Similar results to those presented in Table 7 were obtainedwith a composition comprising 40% Ensure® prepared and administered inthe same fashion as the 50% cream.

TABLE 7 Delivery of Therapeutically Effective Amounts of CO In VivoConcentration at Assay Specimen Point (% CO-Hb) Neg. Control    0.8 ±1.9% 2 3.0 ± 1.1 3 4.3 ± 0.6

The present Example thus shows that compositions according to thepresent invention can be successfully used preclinically in vivo todeliver therapeutically-effective amounts of CO to the blood ofsubjects.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification be considered asexemplary only, with the true scope of the invention indicated by thefollowing claims.

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1. A method of treating a subject suffering from or at risk ofdeveloping a disease that is treatable with carbon monoxide (CO), saidmethod comprising: administering to the subject a liquid compositioncomprising dissolved gaseous, or entrapped gaseous bubbles of, CO in anamount of 85 mg/l or more.
 2. The method of claim 1, wherein the liquidcomposition comprises dissolved gaseous, or entrapped gaseous bubblesof, CO in an amount of from 85 mg/l to 400 mg/l.
 3. The method of claim1, wherein the volume of the liquid composition administered per day is2 liters or less.
 4. The method of claim 1, wherein the volume of theliquid composition administered per day is 1 liter or less.
 5. Themethod of claim 1, wherein the administration is by way of thegastrointestinal tract.
 6. The method of claim 5, wherein administrationby way of the gastrointestinal tract is by oral ingestion.
 7. The methodof claim 5, wherein administration by way of the gastrointestinal tractis by rectal administration.
 8. The method of claim 1, wherein theadministration is intravenous or subcutaneous.
 9. The method of claim 1,wherein the disease is: a hematological disease, an inflammatorydisease, an ischemic disease, sepsis, a cardiovascular disease, kidneydisease, liver disease, or a gastrointestinal disease.
 10. The method ofclaim 9 wherein the ischemic disease is related to ischemia/reperfusioninjury.
 11. The method of claim 9, wherein the hematological disease iscaused by an abnormal hemoglobin.
 12. The method of claim 9, wherein thedisease is Sickle Cell Disease, Hemoglobin C Disease, Hemoglobin SCDisease, or Hemoglobin S beta-thalassemia.
 13. The method of claim 1,wherein the disease is related to solid organ transplantation.
 14. Themethod of claim 1, wherein the disease is related to a burn injury. 15.The method of claim 1, wherein administering is repeated one or moretimes to provide a treatment regimen.
 16. The method of claim 1, whereinthe dissolved gaseous, or entrapped gaseous bubbles of, CO is present inan amount sufficient to prevent or treat at least one clinical symptomof a disease or disorder affected by CO.
 17. The method of claim 1,wherein the liquid composition further comprises at least one of:protein, lipid, fat, triglyceride, complex carbohydrate, sugar,artificial sugar substitute, fruit juice, carbohydrate, cellulose,fiber, citric acid, artificial flavoring, natural flavoring, gum,pectin, ascorbic acid, preservative, saponin, oil, oil emulsion, and asalt.
 18. The method of claim 1, wherein the liquid comprises water,ethanol, or both.
 19. The method of claim 1, wherein the liquidcomposition is a liquid colloidal dispersion, liquid suspension,emulsion, or foam.
 20. The method of claim 1, wherein the entrapped COgas bubbles are present within the liquid composition when the liquidcomposition is at an ambient pressure environment.